Replication fork stalling and collapse is a major source of genomic instability and subsequent neoplasia. Such stressed forks can be conservatively repaired and restarted using homologous recombination (HR) repair, initiated by generating an endogenous nick at the fork junction. While there are candidate nucleases for generating this DSB, most mammalian stressed forks can be restarted without these nucleases, and the origin of this nick remains undefined. This nick permits the 5' end resection that initiate HR and prohibits non- homologous end-joining (NHEJ), the pathway leading to genomic instability during replication stress. We found that the previously uncharacterized nuclease EEPD1 is an essential initiating step in HR repair of stalled forks. EEPD1 has two amino terminal Helix-hairpin-Helix domains that resemble prokaryotic fork repair component RuvA and a carboxy terminal DNase I-like endonuclease. After replication stress, EEPD1 is recruited to stalled forks and increases nicking. EEPD1 enhances 5' DNA end resection and restart of stalled forks. It is required for proper ATR and CHK1 phosphorylation, and ?-H2Ax, Rad51 and RPA32 foci formation. Consistent with this, purified recombinant EEPD1 protein has unique 5' DNA endonuclease activity which enhances Exo1 nuclease activity at fork structures. Both Exo1 and EEPD1 (T134) are phosphorylated in S/G2 by CDK1. EEPD1 depletion generates nuclear and cytogenetic anomalies, made worse by replication stress. Inhibiting 53BP1 partially rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. Examining the role of EEPD1 in a rapidly proliferating organism, we found that Zebrafish embryos depleted of EEPD1 demonstrate significant nuclear abnormalities, and increased developmental delay and death. These data demonstrate that genomic stability during replication stress is maintained by EEPD1, yet the mechanism by which EEPD1 performs this is not defined. This project will assess how EEPD1 is regulated in 4 aims: 1) What structures of EEPD1 are important for stressed replication fork repair? 2) How does CDK1 phosphorylation of EEPD1 regulate stressed fork repair? 3) What role does EEPD1 play in pathway choice for stressed fork repair? 4) Does EEPD1 mediate response to replication stressing cancer therapy?